This project aims to develop, improve, and exploit new molecules, mostly genetically targetable, for measuring and/or manipulating neuronal messengers and signals such as calcium, glutamate, GABA, action potentials, surface exposure of selected proteins, singlet oxygen, protein-protein proximity over tens of nm, turnover of key synaptic proteins at steady-state vs. conditions of new synapse formation, and activity of extracellular proteases during stroke. Calcium indicators genetically targeted to channel mouths or synaptic proteins will be used to investigate privileged nanodomains of calcium, long postulated but not directly measured. Fluorescent protein (FP)-based indicators of glutamate should enable quantitative dissection of pre- vs. postsynaptic components of excitatory synapse modulation, localization of excitatory signaling, and mechanisms of excitotoxicity. Analogous indicators of gamma-aminobutyrate (GABA) would extend such analyses to inhibitory synapses. Channels that sensitize neurons to fire action potentials in response to red/near-infrared light or magnetic field transients would allow noninvasive transcranial stimulation of neurons in behaving animals. FPs excitable by red/near-infrared would greatly improve detection of gene expression and protein fate deeper inside intact tissues and organisms, due to greater penetration and lesser autofluorescence at long wavelengths. Labeling of extracellular hexahistidine motifs by fluorescent chelators of zinc offers fast detection of the surface exposure of designated proteins, useful for understanding cycling of proteins at synapses and during capacitative calcium entry. FP-based generators and sensors of singlet oxygen should enable assays of protein-protein proximity over distances of many tens of nm, the size of large supramolecular complexes. Turnover of synaptic proteins will be investigated with photoconvertible FPs and two-color pulse-chase labeling of tetracysteine motifs, seeking proteins that are incorporated mainly into nascent synapses and can serve as molecular markers of new synapse formation. Activatable cell-penetrating peptides that accumulate where matrix metalloproteinases are active will be used to probe the role of these enzymes in stroke models. Relevance: New molecular tools will be developed for analyzing key molecules and signals. Such tools will help elucidate mechanisms of neuronal information processing, communication, lifecycles, control of behavior, and pathological injury.